Nanofabrication techniques offer the possibility to create solid state pores or apertures with diameters and lengths similar to diameters and lengths of single nucleotides or proteins. Solid state nanopores permit use of non-physiological conditions for structural manipulation of biopolymers, such as non-neutral pH levels, high temperatures and/or high voltage differences. Use of a solid state substrate will allow a more straightforward manipulation of surface chemistry in the pore, which may be critical to fine-tune the rate of nucleic acid translocation or the degree of ionic current reduction associated with passage of a polymer, such as a poly-nucleotide through a nanopore.
Kasianowicz et al, in “Characterization of individual polynucleotide molecules through a membrane channel,” Proc. Nat. Acad. Sci. vol. 93 (1996) 195-223, have used a pore of diameter about 1.5 nanometers (nm) in the bacterial α-hemolysin ion channel protein, and have applied an electrical field to drive a negatively charged polynucleotide through the pore from one side to the other, which transiently reduces ionic conductance through the pore. Akeson et al, in “Microsecond Time Scale Discrimination Among Polycytidylic Acis in Homopolymers or as Segments Within Single RNA Molecules,” Biophys. Jour. Vol. 77 (1999) 3227-3233, have shown that polynucleotides of different lengths can be discriminated by time duration of translocation as the nucleotide sequence passes through a pore. Translocation of different nucleotide homopolymers reduces ionic conductance of α-hemolysin by characteristic amounts, which suggests that the individual nucleotides in a heteropolymer could be identified, if passed through a nanopore of appropriate dimensions and composition. However, α-hemolysin has a pore length as long as a sequence of about 20 nucleotides so that discrimination between individual nucleotides using α-hemolysin is not possible.
What is needed is a system that provides rapid and accurate identification of ordered components of a nucleic acid, protein or similar polymer, at rates up to and above one component per μsec. Preferably, the approach should adequately discriminate between the different ordered components present in the polymer and provide accurate ordering, with an acceptable error rate that is controllable by varying the rate at which the polymer components pass through and is read by the system.